736 J. AMER. SOC. HORT. SCI. 128(5):736–740. 2003.

J. AMER. SOC. HORT. SCI. 128(5):736–740. 2003.

Simultaneous Visualization of 5S and 45S rDNAs in Persimmon (Diospyros kaki) and Several Wild Relatives (Diospyros spp.) by Fluorescent in situ Hybridization (FISH) and MultiColor FISH (MCFISH)Young A Choi,1 Ryutaro Tao, Keizo Yonemori, and Akira SugiuraLab of Pomology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

ADDITIONAL INDEX WORDS. chromosome marker, physical mapping, repetitive DNA, ribosomal RNA genes, rDNA

ABSTRACT. 5S ribosomal DNA (rDNA) was visualized on the somatic metaphase chromosome of persimmon (Diospyros kaki) and ten wild Diospyros species by fl uorescent in situ hybridization (FISH). The digoxigenin (DIG)-labeled 5S rDNA probe was hybridized onto the chromosomes and visualized by incubation with anti-DIG-fl uorescein isothiocyanate (FITC). Strong signals of 5S rDNA probe were observed on several chromosomes of Diospyros species tested. Furthermore, multicolor FISH using 5S and 45S rDNA probes differently labeled with DIG and biotin, revealed separate localization of the two rDNA genes on different chromosomes of Diospyros species tested, suggesting that 5S and 45S rDNA sites can be used as chromosome markers in Diospyros. The number of 5S rDNA sites varied with the Diospyros species. More 5S rDNA sites were observed in four diploid species native to Southern Africa than in three Asian diploid species. The former had four or six 5S rDNA sites while the latter had two. Three Asian polyploidy species had four to eight 5S rDNA sites. Among the Asian species, the number of 5S rDNA sites seemed to increase according to ploidy level of species. These features of 5S rDNA sites were very similar to those of 45S rDNA sites in Diospyros. Phylogenetic relationship between D. kaki and wild species tested are discussed based on the number and chromosomal distribution of 5S and 45S rDNA.

Persimmon (Diospyros kaki Thunb.) is one of the most im-portant fruit trees cultivated for centuries in Japan (Sugiura and Subhadrabandhu, 1996). This species is also distributed in tem-perate regions of east Asia and at least 1000 cultivars have been developed. The genus Diospyros, to which persimmon belongs, consists of 400-500 species (Yonemori et al., 2000). Wild spe-cies of Diospyros are mostly diploid (2n = 2x = 30) with a small number of tetraploid species (2n = 4x = 60), while cultivated species, such as D. kaki, are hexaploid (2n = 6x = 90). Some of the seedless cultivars of D. kaki have been reported as nonaploid (2n = 9x = 135) (Tamura et al., 1998; Zhuang et al., 1990). Thus, it is speculated that a single or several diploid and/or tetraploid wild species were involved in the speciation of the cultivated polyploid Diospyros species.

So far, the relationship between wild and cultivated Diospyros species has been discussed based on the limited information ob-tained from isozyme, mitochondrial DNA and chloroplast DNA analyses (Nakamura and Kobayashi, 1994; Tao and Sugiura, 1987; Yonemori et al., 1998). Little information is available about the speciation of cultivated hexaploid species and the phylogenetic relationships among Diospyros species. Although chromosome numbers and nuclear DNA contents for Diospyros species have been reported (Tamura et al., 1998; Zhuang et al., 1990), only a few cytological studies have been conducted for the Diospyros. Small chromosomes of Diospyros (2-3 µm long on the average at metaphase) make observation with a light microscope diffi cult (Choi et al., 2002; Tamura et al., 1998).

Fluorescent in situ hybridization (FISH), using labeled specifi c DNA fragments as probes was developed in the late 1980s as a most effective plant cytogenetic technique (Jiang and Gill, 1994). Direct visualization of defi ned DNA sequences such as ribosomal

Received for publication 30 Dec. 2002. Accepted for publication 13 May 2003. We thank Yoshio Sano, Faculty of Agriculture, Hokkaido University, Kiichi Fukui and Takefumi Sone, Department of Bioengineering, Osaka University, for their courtesy of materials, helpful discussions and suggestions.1Corresponding author; e-mail choi@kais.kyoto-u.ac.jp.

RNA gene (rDNA) on the chromosomes has been applied to study the phylogenetic relationship in horticultural plants such as Al-lium and Brassica (Hasterok et al., 2001; Richroch et al., 1992). Furthermore, multicolor FISH (MCFISH) using 5S and 45S rDNA specifi c probes simultaneously have provided valuable information on the evolution of rDNA sites and the relationships between wild and cultivated polyploid species (Mishima et al., 2002; Raina and Mukai, 1999; Schrader et al., 2000; Taketa et al., 1999). Recently, FISH using a 45S rDNA probe was found useful to elucidate the chromosomal location and the variation in the number of sites of 45S rDNA in 10 Diospyros species (Choi et al., 2003).

In the present study, we performed FISH for physical mapping of 5S rDNA and MCFISH for simultaneous visualization of 5S and 45S rDNA genes on the somatic metaphase chromosomes of D. kaki and wild Diospyros species. The phylogeny of Diospyros species tested is discussed with special reference to variation in the numbers and chromosomal location of 5S rDNA sites compared with those of 45S rDNA.

Materials and Methods

PLANT MATERIALS AND CHROMOSOME PREPARATION. Diospyros species used in this study are shown in Table 1 with their ploidy levels and regional distributions. Young roots were collected from rooted shoots in vitro or seedlings as described before (Choi et al., 2002). Root tips (1-2 cm long) were pretreated with 2 mM 8-hydroquinoline solution for 5 h at 4 °C and fi xed in a methanol-acetic acid (3:1) solution. Chromosome samples were prepared by an enzymatic maceration and air drying method (Fukui, 1996). The enzyme solution was composed of 4% (w/v) cellulase RS (Yakult Honsha, Tokyo), 1% (w/v) pectolyase Y-23 (Kikkoman Co., Tokyo), 0.07 M KCl and 7.5 mM Na2EDTA (pH 4.0).

5S rDNA PROBE PREPARATION. For 5S rDNA detection, the 5S rDNA coding region was PCR amplifi ed from genomic DNA of Diospyros kaki using oligonucleotide primers designed by Fukui et al. (1994a): a forward primer 5S-F (5ʼ- GGATGCGATCATAC-

9391-MolB 736 7/10/03, 11:04:55 AM

737J. AMER. SOC. HORT. SCI. 128(5):736–740. 2003.

CAGCAC -3ʼ) and a reverse primer 5S-R (5ʼ-GGGAATGCAA-CACGAGGACT-3ʼ). A standard PCR method was carried out using a thermal cycler (Perkin Elmer Cetus, Norwalk, Conn.). Total DNA of D. kaki isolated by the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987) was used as a template DNA. For PCR, a 50-µL aliquot reaction mixture containing 50 ng of template DNA, 0.2 mM of dNTP mixture, 0.4 µM of each primer and 1 unit of Taq DNA polymerase (Takara Shuzo Co., Ltd., Tokyo, Japan) was used. Samples were amplifi ed by 35 thermal cycles of 94 °C for 30 s, 58 °C for 1.5 min, and 72 °C for 2 min. The PCR product was cloned into a TA cloning vector (Promega, Tokyo, Japan). Nucleotide sequences of the inserts of several clones were determined using an automatic DNA sequencer (ABI PRISM, Applied Biosystems, Calif.). The inserts of the cloned DNA were amplifi ed and labeled with digoxigenin (DIG)-11-dUTP (Roche, Mannheim, Germany) simultaneously by PCR using 5S-F and 5S-R primers as described by Fukui et al. (1994a).

45S rDNA PROBE PREPARATION. 45S rDNA, consisting of a 18S-5.8S-25S rDNA gene cluster of rice (Sano and Sano, 1990) was labeled with biotin-16-dUTP by the High Prime DNA la-beling system according to the supplier's instructions (Roche, Mannheim, Germany).

FISH/MCFISH. Before hybridization, the chromosome prepa-rations were incubated with 100 mg·mL–1 RNase A and washed with 2× SSC (0.3 M NaCl and 0.03 M sodium citrate), dehydrated with a 70%, 95%, and 99% ethanol series.

The FISH procedure using 5S rDNA specifi c probe was the same as that described by Fukui et al. (1994a). The MCFISH procedure using DIG-labeled 5S and biotinylated 45S rDNA probes was performed as described by Taketa et al. (1999) with a slight modifi cation of probe concentration and its composition as follows. The probe mixture consisted of 20 ng of both labeled probes per slide that were dissolved in 15 µL mixture of 50% formamide in 2× SSC. The biotinylated probe was detected with avidin-rhodamine (1%, v/v, Roche, Mannheim, Germany) and biotinylated anti-avidin (1%, w/v, Vector Laboratories, Burlin-game, Calif.) was applied as the secondary amplifi cation of the fl uorescent signals. The DIG labeled probe was detected with anti-DIG-fl uorescein isothiocyanate (FITC) (10%, w/v, Roche, Mannheim, Germany) at the step of secondary amplifi cation of biotinylated probe for simultaneous detection of both probes. The chromosomes on the slides were counterstained with 0.5 µg·mL–1 4, 6-diamidino-2-phenylindole (DAPI) and mounted with an antifadant solution as described by Choi et al. (2002). The chromosome samples were observed with a fl uorescent microscope (Axiophot, Zeiss, Oberkochen, Germany) with a

high-sensitivity cooled CCD camera (PXL 1400, Photometrics, Ariz.). The B- and G- light excitation fi lters were used for the detection of FITC and rhodamine, respectively. The signal im-ages were analyzed by imaging software (IPLab Spectrum 3.1, Signal Analytics, Calif.). More than 30 to 50 cells from at least ten metaphase slides in each species were observed for FISH and MCFISH.

Results

5S rDNA SITES IN ELEVEN DIOSPYROS SPECIES. Figure 1 shows the nucleotide sequence corresponding to the coding region of 5S rDNA cloned from D. kaki. The fragment of 5S rDNA from D. kaki showed higher than 97% DNA sequence identity to that from tomato, tobacco and Arabidopsis thaliana.

FISH using DIG-labeled 5S rDNA as a probe was performed on somatic chromosomes in 11 Diospyros species with different ploidy levels. The 5S rDNA specifi c probe hybridized to various numbers of sites ranging from two to six in the diploid species. Diospyros glabura, native to southern Africa, showed four 5S rDNA-specifi c signals on the chromosomes (Fig. 2A), and the other southern African species, D. austroafricana, D. lycioides and D. simii, appeared to have six 5S rDNA sites (Fig. 2B-D). Four Asian diploid species, D. ehretioides, D. morrisiana, D. oldhami and D. lotus, carried two 5S rDNA sites on the short arms of chromosomes (Fig. 2E–H). A variation in signal intensity was frequently observed in D. lotus (Fig. 2H and Fig. 3F, arrow). Three polyploid species carried four to eight rDNA sites on their chromosomes. Tetraploid D. rhombifolia had four chromosomes carrying 5S rDNA sites on the short arms of chromosomes (Fig. 2I). Hexaploid D. kaki bore eight 5S rDNA sites on its chromo-somes (Fig. 2J). Three of the four pairs of 5S rDNA sites of D. kaki were visualized at the centromeric regions while the other pair was detected from proximal regions on the long arms (Fig 2J, arrows) of chromosomes. Another hexaploid, D. virginiana, bore six 5S rDNA sites, which were visualized on the short arms

Table 1. Ploidy levels and regional distributions of eleven Diospyros species used in this study.

Species Ploidy level DistributionD. glabura 2n = 2x = 30 Subtropical region of southern AfricaD. austroafricana 2n = 2x = 30 Subtropical region of southern AfricaD. lycioidesz 2n = 2x = 30 Subtropical region of southern AfricaD. simii 2n = 2x = 30 Subtropical region of southern AfricaD. ehretioidesz 2n = 2x = 30 Tropical and subtropical region of southeast AsiaD. morrisiana 2n = 2x = 30 Subtropical and temperate region of east AsiaD. oldhami 2n = 2x = 30 Subtropical and temperate region of east AsiaD. lotus ‘Kunsenshi ̓ 2n = 2x = 30 Temperate region of central and east AsiaD. rhombifolia 2n = 4x = 60 Temperate region of east AsiaD. kaki ‘Jiro ̓ 2n = 6x = 90 Temperate region of east AsiaD. virginiana 2n = 6x = 90 Temperate region of north AmericazSpecies used only in single mapping of 5S rDNA loci in this study.

Fig. 1. The nucleotide sequences corresponding to the coding region of 5S rDNA cloned from Diospyros kaki. Arrows indicate the sequence of oligonucleotide primers used in PCR amplifi cation of 5S rDNA sequence: F = the sequence of a forward primer; R = the reversed sequence of a reverse primer.

9391-MolB 737 7/10/03, 11:05:00 AM

738 J. AMER. SOC. HORT. SCI. 128(5):736–740. 2003.

of chromosomes (Fig. 2K). All green signals from fl uorescein isothiocyanate (FITC) were detected from centromeric or proximal regions on separate chromosomes of Diospyros species tested in this study.

SIMULTANEOUS VISUALIZATION OF 5S AND 45S rDNA GENES ON THE CHROMOSOMES OF DIOSPYROS SPECIES BY MCFISH. MCFISH, performed using DIG-labeled 5S rDNA and biotin-labeled 45S rDNA probes, visualized 5S rDNA (green signals detected with anti-DIG-FITC) and 45S rDNA (red signals detected with avidin-rhodamine) sites simultaneously on the chromosomes of the nine

Diospyros species (Fig. 3A–I). The 5S and 45S rDNA sites were separately localized on different chromosomes in all species used in this study.

Discussion

Probes specifi c to 45S rDNA and its subunits (5.8S, 18S or 25S) have been re-ported as effective chromosome markers in fruit trees (Roose et al., 1998; Schuster et al., 1997; Yamamoto et al., 1999) including Diospyros species (Choi et al., 2003). Since the 5S rDNA gene can be another useful chromosome marker (Cuadrado et al., 1995; Hasterok et al., 2001; Kamisugi et al., 1994; Mukai et al., 1990; Roose et al., 1998; Schuster et al., 1997), we determined if the 5S rDNA specifi c probe could be a chromosome marker for Diospyros species in this study. The signal intensity was strong enough to be detected and the probe hybridized to several chromo-somes suggesting that the 5S rDNA specifi c probe is an effective chromosome marker in Diospyros. Hence, the 5S rDNA gene, an especially effective marker for diploid spe-cies, was clearly visualized by FISH even in the hexaploid species, such as D. kaki and D. virginiana.

Variations in the number of 5S rDNA sites of Diospyros species tested appeared to be

Fig. 2. Visualization of 5S rDNA loci on chromosomes stained with 4, 6-diamidino-2-phenylindole (DAPI). Color images were obtained by combining the pseudo green fl uorescein isothiocyanate (FITC) signals from 5S rDNA loci and the pseudo blue signals from chromosomes stained with DAPI. (A) Diospyros glabura (2n = 2x = 30), (B) D. austroafricana (2n = 2x = 30), (C) D. lycioides (2n = 2x = 30), (D) D. simii (2n = 2x = 30), (E) D. ehretioides (2n = 2x = 30), (F) D. morrisiana (2n = 2x = 30), (G) D. oldhami (2n = 2x = 30), (H) D. lotus (2n = 2x = 30), (I) D. rhombifolia (2n = 4x = 60), (J) D. kaki (2n = 6x = 90), (K) D. virginiana (2n = 6x = 90). Arrow in H indicates variation in signal intensity of 5S rDNA repeat unit observed in D. lotus. Arrows in J indicate proximal localization of 5S rDNA on chromosomes of D. kaki. Bar represents 10 µm.

quite similar to those of 45S rDNA sites (Table 2; Choi et al., 2003). The number of 5S rDNA sites in diploid species varied from two to six whereas that of 45S rDNA sites from two to eight. Four species, native to South Africa, had more 5S and 45S rDNA sites than those of Asian diploids. The geographical variability in the number of rDNA sites was observed in other plant species such as Oryza and Zamiaceae (Fukui et al., 1994b; Tagashira and Kondo, 2001). The recent study on the sequence analysis of the ITS and matK regions of Diospyros species revealed that African species were phylogenetically most distant species from D. kaki among the species tested (unpublished laboratory data). The difference in

the number of 5S rDNA sites between African and Asian species as observed in 45S rDNA sites suggests that the southern African species have evolved forming an independent phylogenetic group in Diospyros. This could suggest that southern African species were not involved directly in the speciation of cultivated hexaploid species. Furthermore, within the Asian species, the numbers of 5S rDNA sites seemed to increase depending on the ploidy levels of species as did the number of 45S rDNA sites (Table 2). This result was different from many other plant species that did not show any correlation between the number of 5S and 45S rDNAs sites (Castilho and Heslop-Harrison, 1995; Mishima et al., 2002; Schrader et al., 2000; Taketa et al., 1999).

Since the chromosomal distribution of rDNA genes could be an additionally important indicator in the phylogenetic study among related plant species (Castilho and Heslop-Harrison, 1995; Maluszynska and Heslop-Harrison, 1993; Zhang and Sang, 1999), we mapped 5S and 45S rDNA sites of nine Dios-pyros species simultaneously by MCFISH (Fig. 3). In all nine species tested, 45S rDNA was located at the nucleolus organizer region (NOR) or other chromosomal regions while 5S rDNA was detected at the proximal or centromeric parts of chromosomes

9391-MolB 738 7/10/03, 11:05:05 AM

739J. AMER. SOC. HORT. SCI. 128(5):736–740. 2003.

number of the target site in the chromosomes (Leitch and Heslop-Harrison, 1992). This length variation of 5S rDNA sites observed in D. lotus presumably originated from major deletion-insertion events in the region of the 5S rDNA repeat units during the evolution of spe-cies as reported in rye and tobacco (Cuadrado et al., 1995; Kitamura et al., 2000).

In conclusion, FISH and MCFISH using the 5S and 45S rDNA probes, successfully visualized their sites on the chromosomes of Diospyros. The pattern of localization of 5S and 45S rDNA genes on the chromosomes would serve as effective chromosome mark-ers for Diospyros species. Furthermore, the similarity in pattern of variation in the number of 5S and 45S rDNA sites tested may have a signifi cant implication for elucidating the phylogenetic relationship and the speciation

of polyploid species of Diospyros.

Literature Cited

Castilho, A. and J.S. Heslop-Harrison. 1995. Physical mapping of 5S and 18S-25S and repetitive DNA sequences in Aegilops umbellulata. Genome 38:91–96.

Choi, Y.A., R. Tao, K. Yonemori, and A. Sugiura. 2002. Multicolor genomic in situ hybridization identifi es parental chromosomes in somatic hybrids of Diospyros kaki and D. glandulosa. HortScience 37:184–186.

Choi, Y.A., R. Tao, K. Yonemori, and A. Sugiura. 2003. Physical map-ping of 45SrDNA by fl uorescent in situ hybridization in Diospyros kaki (persimmon) and its wild relatives. J. Hort. Sci. Biotechnol. 78:265–271.

Cuadrado, A., N. Jouve, and J.S. Heslop-Harrison. 1995. Physical mapping of the 5S rRNA multigene family in 6x triticale and rye: Identifi cation of a new rye locus. Genome 38:623–626.

Doyle, J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bul. 19:11–15.

Fukui, K. 1996. Plant chromosomes at mitosis, p. 1–17. In: K. Fukui and S. Nakayama (eds.). Plant chromosomes: Laboratory methods. CRC Press, BocaRaton, Fla.

Fukui, K., Y. Kamisugi, and F. Sakai. 1994a. Physical mapping of 5S rDNA loci by direct-cloned biotinylated probes in barley chromosomes. Genome 37:105–111.

Fukui, K., N. Ohmido, and G.S. Khush. 1994b. Variability in rDNA loci in the genus Oryza detected through fl uorescence in situ hybridization. Theor. Appl. Genet. 87:893–899.

Hasterok, R. and J. Maluszynska. 2000. Nucleolar dominance does not occur in root tip cells of allotetraploid Brasicca species. Genome 43:574–579.

Hasterok, R., G. Jenkins, T. Langdon, R.N. Jones, and J. Maluszynska. 2001. Ribosomal DNA is an effective marker of Brasicca chromosomes. Theor. Appl. Genet. 103:486–490.

Jiang, J. and B.S. Gill. 1994. Nonisotopic in situ hybridization and plant

Fig. 3. Simultaneous visualization of 5S rDNA and 45S rDNA sites. Color images were obtained by combining the pseudo green FITC signals from 5S rDNA sites, the pseudo red rhodamine signals from 45S rDNA sites and the pseudo blue signals from chromosomes stained with DAPI. (A) Diospyros glabura (2n = 2x = 30), (B) D. austroafricana (2n = 2x = 30), (C) D. simii (2n = 2x = 30), (D) D. morrisiana (2n = 2x = 30), (E) D. oldhami (2n = 2x = 30), (F) D. lotus (2n = 2x = 30), (G) D. rhombifolia (2n = 4x = 60), (H) D. kaki (2n = 6x = 90), (I) D. virginiana (2n = 6x = 90). Arrow in F indicates variation in signal intensity of 5S rDNA repeat unit observed in D. lotus. Bar represents 10 µm.

Table 2. The number of 5S and 45S rDNA sites of eleven Diospyros species used in this study.

Species No. of No. of(ploidy level) 5S rDNA site 45S rDNA sitez

D. glabura (2x) 4 6D. austroafricana (2x) 6 6y

D. lycioides (2x) 6 8D. simii (2x) 6 8D. ehretioides (2x) 2 2D. morrisiana (2x) 2 2D. oldhami (2x) 2 2D. lotus ‘Kunsenshi ̓(2x) 2 4D. rhombifolia (4x) 4 8D. kaki ‘Jiro ̓(6x) 8 8D. virginiana (6x) 6 8zReported previously by Choi et al., 2003.yThe number of 45S rDNA sites determined in this study.

other than those bearing the NOR. The different multiloci chro-mosomal distributions of the two rDNA genes showed that they are independent of each other as indicated in many other plants (Cuadrado et al., 1995; Kamisugi et al., 1994; Leitch and Heslop-Harrison, 1992; Rogers and Bendich, 1987). The localization of 5S and 45S rDNA genes on different chromosomes of Diospyros species tested suggests that reorganization has rarely occurred between the two rDNAs sites as indicated in Brasicca (Hasterok and Maluszynska, 2000).

As shown in Fig. 2H (arrow), a variation in signal intensity between 5S rDNA sites was frequently observed in D. lotus. Although in situ hybridization is not a quantitative method, dif-ferences in signal strength are correlated with variation in copy

9391-MolB 739 7/10/03, 11:05:12 AM

740 J. AMER. SOC. HORT. SCI. 128(5):736–740. 2003.

genome mapping: The fi rst 10 years. Genome 37:717–725.Kamisugi, Y., S. Nakayama, R. Nakajima, H. Ohtsubo, E. Ohtsubo, and

K. Fukui. 1994. Physical mapping of the 5S ribosomal RNA genes on rice chromosome 11. Mol. Gen. Genet. 245:133–138.

Kitamura, S., M. Inoue, N. Ohmido, and K. Fukui. 2000. Quantitative chromosome maps and rDNA localization in the T subgenome of Ni-cotiana tabacum L. and its putative progenitors. Theor. Appl. Genet. 8:1180–1188.

Leitch, I.J. and J.S. Heslop-Harrison. 1992. Physical mapping of the 18S-5.8S-26S rRNA genes in barley by in situ hybridization. Genome 35:1013–1018.

Maluszynska, J. and J.S. Heslop-Harrison. 1993. Physical mapping of rDNA loci in Brassica species. Genome 36:774–781.

Mishima, M., N. Ohmido, K. Fukui, and T. Yahara. 2002. Trends in site number change of rDNA loci during polyploid evolution in Sanguisorba (Rosaceae). Chromosoma 110:550–558.

Mukai, Y., T.R. Endo, and B.S. Gill. 1990. Physical mapping of the 5S rRNAmultigene family in common wheat. J. Heredity 81:290–295.

Nakamura, Y. and S. Kobayashi. 1994. DNA restriction fragment length variability in Diospyros kaki and related Diospyros species. HortScience 29:809–811.

Raina, S.N. and Y. Mukai. 1999. Detection of a variable number of 18S-5.8S-26S and 5S ribosomal DNA loci by fl uorescent in situ hybridization in diploid and tetraploid Arachis species. Genome 42:52–59.

Richroch, A., E.B. Peffl ey, and R.J. Baker. 1992. Chromosomal location of rDNA in Allium: In situ hybridization using biotin- and fl uorescein- labelled probe. Theor. Appl. Genet. 83:413–418.

Rogers, S.O. and A.J. Bendich. 1987. Ribosomal RNA genes in plants: variability in copy number and in the intergenic spacer. Plant Mol. Biol. 9:509–520.

Roose, M.L., T. Schwarzacher, and J.S. Heslop-Harrison. 1998. The chromosomes of Citrus and Poncirus species and hybrids: Identifi ca-tion of characteristic chromosomes and physical mapping of rDNA loci using in situ hybridization and fl uorochrome banding. J. Hered. 89:83–86.

Sano, Y. and R. Sano. 1990. Variation of the intergenic spacer region of ribosomal DNA in cultivated and wild rice species. Genome 33:209–218.

Schrader, O., H. Budahn, and R. Ahne. 2000. Detection of 5S and 25S rRNA genes in Sinapis alba, Raphanus sativus and Brassica napus by double fl uorescence in situ hybridization. Theor. Appl. Genet. 100:665–669.

Schuster, M., J. Fuchs, and I. Schubert. 1997. Cytogenetics in fruit breed-ing localization of ribosomal RNA genes on chromosomes of apple (Malus domestica Borkh.) Theor. Appl. Genet. 94:322–324.

Sugiura, A. and S. Subhadrabandhu. 1996. Overview of persimmon culture. Chronica Hort. 36:14–15.

Tagashira, N. and K. Kondo. 2001. Chromosomes phylogeny of Zamia and Ceratozamia by means of Robertsonian changes detected by fl uorescence in situ hybridization (FISH) technique of rDNA. Plant Syst. Evol. 227:145–155.

Taketa, S., G.E. Harrison, and J.S. Heslop-Harrison. 1999. Comparative physical mapping of the 5S and 18-25S rDNA in nine wild Hordeum species and cytotypes. Theor. Appl. Genet. 98:1–9.

Tamura, M., R. Tao, K. Yonemori, N. Utsunomiya, and A. Sugiura. 1998. Ploidy level and genome size of several Dioispyros species. J. Jpn. Soc. Hort. Sci. 67:306–312.

Tao, R. and A. Sugiura. 1987. Cultivar identifi cation of Japanese persim-mon by leaf isozymes. HortScience 22:932–935.

Yamamoto, M., T. Shimada, T. Haji, N. Mase, and Y. Sato. 1999. Physical mapping of the 18S ribosomal RNA gene of peach (Prunus persica (L.) Batsch) chromosome by fl uorescent in situ hybridization. Breed-ing Sci. 49:49–51.

Yonemori, K., S. Kanzaki, D.E. Parfi tt, N. Utsunomiya, S. Subhadrab-andhu, and A. Sugiura. 1998. Phylogenetic relationship of Diospyros kaki (persimmon) to Diospyros spp. (Ebenaceae) of Thailand and four temperate zone Diospyros spp. from an analysis of RFLP variation in amplifi ed cpDNA. Genome 41:173–182.

Yonemori, K., A. Sugiura, and M. Yamada. 2000. Persimmon genetics and breeding. Plant Breeding Rev.19:191–225.

Zhang, D. and T. Sang. 1999. Physical mapping of ribosomal RNA genes in peonies (Paeonia, Paeoniaceae) by fl uoresecent in situ hybridization: Implication for phylogeny and concerted evolution. Amer. J. Botany. 86:735–740.

Zhuang, D.H., A. Kitajima, M. Ishida, and Y. Sobajima. 1990. Chromosome numbers of Diopyros kaki cultivars. J. Jpn. Soc. Hort. Sci. 59:289–297.

9391-MolB 740 7/10/03, 11:05:19 AM